Potential applicability of cytokines as biomarkers of disease activity in rheumatoid arthritis: Enzyme-linked immunosorbent spot assay-based evaluation of TNF-α, IL-1β, IL-10 and IL-17A

Biomarkers play a pivotal role in the management of rheumatoid arthritis (RA) by facilitating early diagnosis and ‘treat to the target.’ However, no gold standard biomarker has been identified for monitoring the disease activity in RA. Cytokines, a diverse group of small protein molecules secreted by peripheral blood mononuclear cells (PBMCs), play a pivotal role in pathogenesis and disease progression in RA. Research is currently underway to find out the applicability of cytokines as biomarkers in RA. This study aimed to quantify the PBMCs that secrete four types of cytokines; TNF-α, IL-1β, IL-10 and IL-17A in two cohorts of active RA patients (early RA patients and established RA patients), compared to healthy controls (HC), using the enzyme-linked immunosorbent spot (ELISPOT) assay, and to assess their association with measures of disease activity of RA. Patients were recruited from outpatient rheumatology clinics, and the disease activity was assessed using single and composite measures of disease activity. The cytokine expression was evaluated using freshly separated PBMCs from whole blood of RA patients using the ELISPOT assay. The number of PBMCs (counted as spot-forming cells (SFCs) per 105 PBMCs) that secreted the cytokine of interest were statistically significantly higher in early RA patients, compared to HC, for IL-17A (P<0.05). Such an increased number of SFCs was not observed in the established RA group, compared to controls, for any of the cytokines tested. The correlation analysis showed that IL-17A is having a moderate correlation (Spearman`s ρ, p <0.05) with five clinical measures of disease activity, including disease activity score 28 (DAS28). According to the multivariable linear regression models, IL17A was a good predictor of both the disease activity score 28 (DAS28) and clinical disease activity index (CDAI). In conclusion, IL-17A has potential applicability as a biomarker of disease activity of RA.Scopu

Validity of clinical disease activity index (CDAI) to evaluate the disease activity of rheumatoid arthritis patients in Sri Lanka: A prospective follow up study based on newly diagnosed patients

Routine use of the Disease Activity Score-28 (DAS28) to assess the disease activity in rheumatoid arthritis (RA) is limited due to its dependency on laboratory investigations and the complex calculations involved. In contrast, the clinical disease activity index (CDAI) is simple to calculate, which makes the "treat to target" strategy for the management of RA more practical. We aimed to assess the validity of CDAI compared to DAS28 in RA patients in Sri Lanka. A total of 103 newly diagnosed RA patients were recruited, and their disease activity was calculated using DAS 28 and CDAI during the first visit to the clinic (0 months) and reassessed at 4 and 9 months of follow-up visits. The validity of the CDAI, compared to DAS 28, was evaluated. Patients had a female preponderance (6:1) and a short symptom duration (mean = 6.33 months). Internal consistency reliability of CDAI, as assessed by Cronbach’s α test, was 0.868. Convergent validity was assessed by correlation and Kappa statistics. Strong positive correlations were observed between CDAI and DAS 28 at the baseline (0 months), 4 and 9 months of evaluation (Spearman’s r = 0.935, 0.935, 0.910, respectively). Moderate-good inter-rater agreements between the DAS-28 and CDAI were observed (Weighted kappa of 0.660, 0.519, and 0.741 at 0, 4, and 9 months respectively). Discriminant validity, as assessed by ROC curves at 0, 4th, and 9th months of the evaluation, showed the area under the curve (AUC) of 0.958, 0.979, and 0.910, respectively. The suggested cut-off points for different CDAI disease activity categories according to ROC curves were ≤ 4 (Remission), > 4 to ≤ 6 (low), > 6 to ≤ 18 (moderate), > 18 (high). These findings indicate that the CDAI has good concordance with DAS 28 in assessing the disease activity in RA patients, in this study sample

Interferon-beta treatment in multiple sclerosis : Analysis of neutralizing antibodies

Multiple sclerosis (MS) is a disabling chronic neurological disease with a significant impact on patients lives. There is no cure for MS, but recombinant interferon beta (IFN-beta) is currently the most established disease modifying therapy. One of the major clinical problems of IFN-beta is the development of neutralizing antibodies (NAbs) that interfere with the clinical efficacy of the drug. Still, in the medical community, there is some skepticism regarding the clinical use of NAb testing and its interpretation at the individual level. The overall aim of this thesis was to provide evidence-based knowledge which will be useful in clinical management. To some extent, such information could help in understanding similar scenarios for the many emerging biopharmaceuticals that are potentially immunogenic and might induce antibodies which could interfere with clinical efficacy. Biological study materials were blood samples from IFN-beta treated MS patients that were referred from neurology units in Sweden, Norway and Finland to the specialized NAb laboratory in the Neurology Division at the Karolinska University Hospital in Huddinge. NAbs were measured using the MxA induction assay and in vivo IFN-beta bioactivity was measured by the in vivo MxA mRNA assay. We observed an overall 32% of NAb positivity in MS patients who were tested for NAbs from 2003 to 2006. Weekly low-protein-dose intramuscular IFN-beta was found to be the least seroprevalent and immunogenic product compared to the weekly-high-protein-dose subcutaneous IFN-beta preparations. A titer around 150 TRU/ml marked an important IFN-beta bioactivity level for the NAb titers as a significant in vivo drug response was retained up to this level. Titers above 600 TRU/ml were associated with complete loss of IFN-beta bioactivity. Fluctuation of NAb titers was assessed across the functionally critical titer of 150 TRU/ml and showed that the majority (72%) of patients were stable, especially the two extreme ends of the titer spectrum i.e. negative (less than 10 TRU/ml) and very high titers (>600 TRU/ml). As expected, titers close to150 TRU/ml appeared more prone to fluctuation across this level, i.e. offering a greater chance ofregaining good IFN-beta bioactivity, or, reversely, a greater risk of losing bioactivity. These observations were not influenced by treatment duration or sampling interval between consecutive NAb tests. We failed to observe an increased NAb positivity when referrals indicated an impression of disease worsening. Conclusion: Here, we studied several clinically relevant aspects of IFN-beta NAbs. We identified differences between the IFN-beta preparations regarding the frequency of NAb induction, which we refer to as seroprevalence, but also regarding titer levels of induced NAbs, which we refer to as immunogenicity. In a population-based sample of MS patients we demonstrated that these parameters vary between IFN-beta preparations. Further, we identified titer levels that are critical for retaining the in vivo bioactivity of IFN-beta, vital for the interpretation of individual NAb tests. We showed that NAb titers are commonly stable with a greater tendency of fluctuation of mid-range titers. Finally, we showed that a clinical impression of worsening is poorly predictive of NAb status, implicating the necessity of regular NAb testing. Although NAb testing and its interpretation remains a complex area, our results have provided clinically useful knowledge of relevance in the management of MS patients treated with IFN-beta

Scatter plot illustrating linear positive correlation between DAS 28 and CDAI assessed at 3 time points (0 = 0 months, 4 = 4 months, 9 = 9 months).

At all three time points of evaluation, CDAI was strongly correlated with DAS 28; ρ = 0.9357 at 0 months (p<0.05), ρ = 0.9354 at 4 months (p<0.05), ρ = 0.9106 at 9 months (p<0.05). CDAI–clinical disease activity, DAS 28- disease activity score 28.</p

Inter-rater agreement between CDAI and DAS 28 at 3-time points of disease activity assessment.

Inter-rater agreement between CDAI and DAS 28 at 3-time points of disease activity assessment.</p

Raw data.

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Box and whisker plots illustrating the changes in the disease activity in the patient cohort as assessed by CDAI (a) and DAS 28 (b).

The median disease activities at different time points were compared by the Kruskal-Wallis test (with Dunn’s post-hoc analysis). The drops in the median disease activity down the timeline were statistically significant (p < 0.05). Asterix indicate that the difference between groups is statistically significant. DAS 28 –disease activity score 28, CDAI- clinical disease activity index, 0, 4, and 9 in the x-axis indicate the time point of evaluation (0 –baseline, 4–4 months, and 9–9 months).</p